The present invention relates to a system and method for identifying and sorting out rare cells from a mixed population of cells. More particularly, the present invention relates to a system and method which incorporate a cell isolation system including an image processor and a micromanipulator for identifying and sorting out rare cells from a mixed population of cells. The present system and method can be used, for example, to identify and isolate very rare fetal cells having unique morphological features, such as trophoblasts, from a maternal blood sample, such fetal cells can then be subjected to a variety of prenatal diagnostic methods.
Fetal tissue, and in particular fetal DNA, is routinely used in prenatal diagnosis and other medical procedures which require an accurate assessment of the genome of the fetus. Currently, fetal tissue is obtained through the use of amniocentesis, chorionic villi sample (CVS), fetoscopy, or cordocentesis. Thompson and Thompson Genetics in Medicine, 5th Edition, W. B. Saunders Co., Phila., 1991, provides further detail describing these techniques.
Thus, in amniocentesis, a sample of amniotic fluid, which contains fetal cells, is transabdominally removed from the mother with a needle coupled to a syringe. Amniocentesis has inherent associated risks. The major risk is induction of miscarriage which is estimated to occur in approximately 0.5% of all amniocentesis procedures. Other risks include maternal infection and physical damage to the fetus.
In CVS, fetal trophoblast tissue is aspirated from the villi area of the chorion transcervically or transabdominally. The rate of fetal loss associated with this method may be as high as 1%.
Cordocentesis provides a method of obtaining fetal blood directly from the umbilical cord with ultrasonic guidance.
Although generally efficient in retrieving fetal tissue, each of these invasive methods carries great risks to both the mother and the fetus.
A variety of fetal cell types such as, for example, platelets, trophoblasts, erythrocytes and leukocytes cross the placental blood barrier and circulate transiently within maternal blood (Schroder, J., J. 1975, Med. Genet. 12:230-242; Douglas G. W. et al. 1959, Am. J. Obstet. Gynec., 78:960-973).
The identification and isolation of such fetal cells from a maternal blood sample provides a highly desirable non-invasive method for acquiring fetal genetic material for prenatal genetic testing. A major drawback to utilizing such fetal cells as a source of fetal tissue is due to their extreme rarity in maternal blood. For example, one recent report estimates that in the case of fetal erythrocytes, a maternal blood sample contains approximately one fetal erythrocytes to 1.times.10.sup.7 -1.times.10.sup.8 maternal cells (Simpson and Elias, 1993, JAMA 270:2357).
Although several detection methods have been made available through recent advances, including polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), the major difficulty in the routine use of maternal blood for prenatal diagnosis is the inability to enrich the small number of fetal cells in a mixture of maternal cells to yield reliable diagnostic results. It will be appreciated in this respect that substantially zero tolerance for maternal DNA containing cells is tolerated.
Thus, due to the extreme rarity of fetal cells in maternal blood, a number of specialized techniques to enrich and/or isolate the fetal cell fraction or the fetal genetic material from maternal blood have been designed.
One approach has been to use enrichment methods such as gradient centrifugations for isolating fetal cells, see, for example, U.S. Pat. No. 5,432,054. Although limited separation can be achieved using such methods, these methods are typically not sensitive enough to effect the isolation of a fetal cell fraction usable for highly reliable genetic testing, e.g., substantially zero tolerance. In addition, the currently used enrichment methods such as gradient centrifugation all result in substantial cell loss, thereby reducing the number of fetal cells available for subsequent analysis or for use with subsequent cell sorting or enrichment techniques.
Another more sensitive approach that has been utilized in an attempt to isolate fetal cells from a maternal blood sample utilizes labeled antibodies specific for a particular fetal cell type. Antibody labeled cells can then be isolated by a variety of methods which depend on the recognition of the antibody label. For example, fetal cell specific antibodies can be used to label fetal cells in order to facilitate separation of these cells from maternal cellular components by flow cytometry (Herzenberg, L. A., et al., Proc. Natl. Acad. Sci. USA 76, 1453-1455 (1979); Iverson, G. M., et al., Prenatal Diagnosis 1, 61-73 (1981); Bianchi, D. W., et al., Prenatal Diagnosis 11, 523-528 (1991) which can utilize fluorescent activation cell sorting (FACS, Herzenberg et al., 1979, Proc. Natl. Aca. Sci. USA 76:1453), magnetic-activated cell sorting (MACS, Ganshirt-Ahlert et al., 1992, Am. J. Obstet. Gynecol. 166:1350) or a combination of both procedures (Ganshirt-Ahlert et al., 1992, Am. J. Hum. Genet. 51:A48). In addition, a combination of gradient centrifugation and flow cytometry methods can also be used to increase the isolation or sorting efficiency.
Although the non-invasive flow cytometry sorting methods provide an alternative to the currently used invasive techniques, limitations inherent to their design limits such methods from being widely practiced.
A major limitation inherent to the flow cytometry techniques arises from the antibodies utilized by such techniques. Such antibodies, although generated cell specific, often cross react with other unwanted cell types which are in far higher concentration in the sample. As a result, such methods are often only sufficient in enriching for fetal cell types and cannot be used for reliable, zero tolerance, fetal cell isolation.
Several publications describe the isolation of fetal cells via micromanipulation.
Tutschek B. et al., "Isolation of fetal cells from transcervical samples by micromanipulation: Molecular conformation of their fetal origin and diagnosis of fetal aneuploidy" Prenatal Diagnosis. Vol. 15: 951-960, 1995, teach the isolation of fetal cells from transcervical samples by micromanipulation to thereby reduce a possibility of co-isolation of maternal cells. However, Tutschek B. et al. fail to teach isolation of rare cells from maternal blood samples.
Takabayashi H. et al. "Development of a non-invasive fetal DNA diagnosis from maternal blood" Prenatal Diagnosis, Vol. 15:74-77, 1995, teach detection and retrieval of Pappenheim stained fetal nucleated erythrocytes from maternal blood samples via micromanipulation. However, Takabayashi H. et al. fail to teach the isolation of trophoblasts from maternal blood, which are much rarer than fetal nucleated erythrocytes, yet much more morphologically distinct. Furthermore, Takabayashi H. et al. fail to teach image processing doing so.
Cueung M. et al. "Prenatal diagnosis of sickle cell anemia and thalassemia by analysis of fetal cells in maternal blood" Nature Genetics, Vol. 14:264-268, 1996, teach the scraping of fetal nucleated erythrocytes stained with an anti-fetal globin antibody from microscopic slides. However, Cueung M. et al. fail to teach the isolation of trophoblasts from maternal blood, which are much rarer than fetal nucleated erythrocytes, yet much more morphologically distinct. Furthermore, Cueung M. et al. fail to teach image processing doing so. In addition, Cueung M. et al. fail to teach morphological identification of the fetal cells, rather Cueung M. et al. teach the use of immunostaining which does not highlight cellular structures, rather highlights cells in a yes or no fashion and in addition may cross react with maternal cells, which where shown to coexpress fetal globins, resulting in co-isolation of maternal cells.
At present, there remains a need for a rapid and reproducible procedure suitable for processing a large volume of whole blood, and which produces highly efficient isolation of fetal cells from maternal blood.
There is thus a widely recognized need for, and it would be highly advantageous to have, a system and method for the isolation of fetal cells from a maternal blood sample devoid of the above limitations and which qualifies with the zero tolerance requirement.